Generation of Maize (Zea mays) Doubled Haploids via Traditional Methods

Commercial maize hybrid production has corroborated the usefulness of producing inbred lines; however, the delivery of new lines has always been a major time constraint in breeding programs. Traditional methods for developing inbred lines typically require 6 to 10 generations of self-pollination to obtain sufficient homozygosity. To bypass the time and costs associated with the development of inbred lines, doubled haploid (DH) systems have been widely adopted in the commercial production of maize. Within just two generations, DH systems can create completely homozygous and homogeneous lines. A typical maize DH system, utilizing anthocyanin markers R1-nj or Pl1 for haploid selection, is described in this protocol

MYO, a Candidate Gene for Haploid Induction in Maize Causes Male Sterility

Doubled haploid technology is highly successful in maize breeding programs and is contingent on the ability of maize inducers to efficiently produce haploids. Knowledge of the genes involved in haploid induction is important for not only developing better maize inducers, but also to create inducers in other crops. The main quantitative trait loci involved in maize haploid induction are qhir1 and qhir8. The gene underlying qhir1 has been discovered and validated by independent research groups. Prior to initiation of this study, the gene associated with qhir8 had yet to be recognized. Therefore, this research focused on characterizing positional candidate genes underlying qhir8. Pursuing this goal, a strong candidate for qhir8, GRMZM2G435294 (MYO), was silenced by RNAi. Analysis of crosses with these heterozygous RNAi-transgenic lines for haploid induction rate revealed that the silencing of MYO significantly enhanced haploid induction rate by an average of 0.6% in the presence of qhir1. Recently, GRMZM2G465053 (ZmDMP) was identified by map-based gene isolation and shown to be responsible for qhir8. While our results suggest that MYO may contribute to haploid induction rate, results were inconsistent and only showing minor increases in haploid induction rate compared to ZmDMP. Instead, reciprocal crosses clearly revealed that the silencing of MYO causes male sterility

Mapping of QTL and Identification of Candidate Genes Conferring Spontaneous Haploid Genome Doubling in Maize (Zea mays L.)

In vivo doubled haploid (DH) technology is widely used in commercial maize (Zea mays L.) breeding. Haploid genome doubling is a critical step in DH breeding. In this study, inbred lines GF1 (0.65), GF3(0.29), and GF5 (0) with high, moderate, and poor spontaneous haploid genome doubling (SHGD), respectively, were selected to develop mapping populations for SHGD. Three QTL, qshgd1, qshgd2, and qshgd3, related to SHGD were identified by selective genotyping. With the exception of qshgd3, the source of haploid genome doubling alleles were derived from GF1. Furthermore, RNA-Seq was conducted to identify putative candidate genes between GF1 and GF5 within the qshgd1 region. A differentially expressed formin-like protein 5 transcript was identified within the qshgd1 region

Improvement and expansion of doubled haploid technology

Doubled haploid (DH) technology offers a shortcut for one of the most time-consuming processes in plant breeding: arriving at homozygous inbred lines which are in turn used for hybrid production. Therefore, DH systems have been widely adopted, highly utilized in commercial production, and viewed as an invaluable tool for those crops that have it available. Spontaneous production of haploids is a rare phenomenon and is not efficient enough to rely on for commercial production. Therefore, discovering a reliable method of haploid production, which is not something many crops have, is essential to utilize DH systems. Maize breeding programs have a reliable method of producing haploids, making this system possible in commercial production. Past studies have recognized several QTL regions that are involved in haploid induction, which may be utilized in efforts to discover underlying genes and create transgenic inducers in other crops. Although DH systems have shaped the image of a modern maize breeding program, there is always an interest in research to further improve upon this tool. The incorporation of embryo culture, for instance, offers several benefits such as a shorted generation time as well as increased genome doubling rates. This thesis contains four chapters that (1) outline the protocol of standard DH systems in maize, (2) explore distinguishing root morphological features that allow for selection of maize haploid embryos via embryo culture, (3) observe doubling rates when colchicine is added to the growth media during embryo culture, and (4) discover the gene responsible for a QTL (qhir8) of haploid induction in maize. Through these studies, it was found that maize haploids can be selected by primary root length within the first three days of growth within DH program utilizing embryo culture. This selection method is efficient and allows for the addition of embryo culture to a non-transgenic DH program. The addition of embryo culture is beneficial because it not only decreases generation time, but also significantly increases doubling rates. Finally, these studies have identified a gene responsible for qhir8 involved with haploid induction in maize. Finding this gene may allow for the creation of transgenic inducers in orphan crops, allowing for the expansion of DH technology to other crops.</p

Generation of Maize (Zea mays) Doubled Haploids via Traditional Methods

Commercial maize hybrid production has corroborated the usefulness of producing inbred lines; however, the delivery of new lines has always been a major time constraint in breeding programs. Traditional methods for developing inbred lines typically require 6 to 10 generations of self-pollination to obtain sufficient homozygosity. To bypass the time and costs associated with the development of inbred lines, doubled haploid (DH) systems have been widely adopted in the commercial production of maize. Within just two generations, DH systems can create completely homozygous and homogeneous lines. A typical maize DH system, utilizing anthocyanin markers R1-nj or Pl1 for haploid selection, is described in this protocol.This is an accepted manuscript of an article published as Vanous, Kimberly, Adam Vanous, Ursula K. Frei, and Thomas Lübberstedt. "Generation of Maize (Zea mays) Doubled Haploids via Traditional Methods." Current Protocols in Plant Biology: 147-157. doi:10.1002/cppb.20050. Posted with permission.</p

MYO, a Candidate Gene for Haploid Induction in Maize Causes Male Sterility

Doubled haploid technology is highly successful in maize breeding programs and is contingent on the ability of maize inducers to efficiently produce haploids. Knowledge of the genes involved in haploid induction is important for not only developing better maize inducers, but also to create inducers in other crops. The main quantitative trait loci involved in maize haploid induction are qhir1 and qhir8. The gene underlying qhir1 has been discovered and validated by independent research groups. Prior to initiation of this study, the gene associated with qhir8 had yet to be recognized. Therefore, this research focused on characterizing positional candidate genes underlying qhir8. Pursuing this goal, a strong candidate for qhir8, GRMZM2G435294 (MYO), was silenced by RNAi. Analysis of crosses with these heterozygous RNAi-transgenic lines for haploid induction rate revealed that the silencing of MYO significantly enhanced haploid induction rate by an average of 0.6% in the presence of qhir1. Recently, GRMZM2G465053 (ZmDMP) was identified by map-based gene isolation and shown to be responsible for qhir8. While our results suggest that MYO may contribute to haploid induction rate, results were inconsistent and only showing minor increases in haploid induction rate compared to ZmDMP. Instead, reciprocal crosses clearly revealed that the silencing of MYO causes male sterility.This article is published as Vanous, K.; Lübberstedt, T.; Ibrahim, R.; Frei, U.K. MYO, a Candidate Gene for Haploid Induction in Maize Causes Male Sterility. Plants 2020, 9, 773. doi: 10.3390/plants9060773.</p

Utilization of Reduced Haploid Vigor for Phenomic Discrimination of Haploid and Diploid Maize Seedlings

Potential benefits of incorporating embryo culture (EC) into a doubled haploid (DH) program, including shortening the breeding cycle and increasing chromosome doubling rates, make the laborious and tedious task of excising embryos worth the effort. Difficulties arise during embryo selection considering the marker gene R1-nj, which is typically used in DH programs, is not expressed in early stages after pollination. Although transgenic approaches have been implemented to bypass this issue, there is so far no known non-transgenic method of selecting haploid embryos. The findings of this study reveal methods of selecting haploid embryos that allow the possibility of incorporating EC into a DH program without using transgenic inducers. The best performing method involves a machine-learning classifier, specifically a support vector machine, which uses primary root lengths and daily growth rates as traits for classification. Selection by this method can be achieved on the third day after germination. By this method, an average false negative rate of 2% and false positive rate of 9% was achieved. Therefore, the methods presented in this research allow efficient and non-transgenic selection of haploid embryos that is simple and effective.This article is published as Vanous, Kimberly, Talukder Zaki Jubery, Ursula K. Frei, Baskar Ganapathysubramanian, and Thomas Lübberstedt. "Utilization of Reduced Haploid Vigor for Phenomic Discrimination of Haploid and Diploid Maize Seedlings." The Plant Phenome Journal 2, no. 1 (2019). DOI: 10.2135/tppj2018.10.0008. Posted with permission.</p

Utilization of Reduced Haploid Vigor for Phenomic Discrimination of Haploid and Diploid Maize Seedlings

Potential benefits of incorporating embryo culture (EC) into a doubled haploid (DH) program, including shortening the breeding cycle and increasing chromosome doubling rates, make the laborious and tedious task of excising embryos worth the effort. Difficulties arise during embryo selection considering the marker gene , which is typically used in DH programs, is not expressed in early stages after pollination. Although transgenic approaches have been implemented to bypass this issue, there is so far no known non-transgenic method of selecting haploid embryos. The findings of this study reveal methods of selecting haploid embryos that allow the possibility of incorporating EC into a DH program without using transgenic inducers. The best performing method involves a machine-learning classifier, specifically a support vector machine, which uses primary root lengths and daily growth rates as traits for classification. Selection by this method can be achieved on the third day after germination. By this method, an average false negative rate of 2% and false positive rate of 9% was achieved. Therefore, the methods presented in this research allow efficient and non-transgenic selection of haploid embryos that is simple and effective

Mapping of QTL and Identification of Candidate Genes Conferring Spontaneous Haploid Genome Doubling in Maize (Zea mays L.)

In vivo doubled haploid (DH) technology is widely used in commercial maize (Zea mays L.) breeding. Haploid genome doubling is a critical step in DH breeding. In this study, inbred lines GF1 (0.65), GF3(0.29), and GF5 (0) with high, moderate, and poor spontaneous haploid genome doubling (SHGD), respectively, were selected to develop mapping populations for SHGD. Three QTL, qshgd1, qshgd2, and qshgd3, related to SHGD were identified by selective genotyping. With the exception of qshgd3, the source of haploid genome doubling alleles were derived from GF1. Furthermore, RNA-Seq was conducted to identify putative candidate genes between GF1 and GF5 within the qshgd1 region. A differentially expressed formin-like protein 5 transcript was identified within the qshgd1 region.This is a manuscript of an article published as Ren, Jiaojiao, Nicholas Boerman, Ruixiang Liu, Penghao Wu, Benjamin Trampe, Kimberly Vanous, Ursula K. Frei, Shaojiang Chen, and Thomas Lübberstedt. "Mapping of QTL and Identification of Candidate Genes Conferring Spontaneous Haploid Genome Doubling in Maize (Zea mays L.)." Plant Science (2019). doi: 10.1016/j.plantsci.2019.110337. Posted with permission.</p